This invention relates to variants of phytases, in particular variants of ascomycete phytases and variants of basidiomycete phytases, the corresponding cloned DNA sequences, a method of producing such phytase variants, and the use thereof for a number of industrial applications.
Phytic acid or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (or for short myo-inositol hexakisphosphate) is the primary source of inositol and the primary storage form of phosphate in plant seeds. Phytin is a mixed potassium, magnesium and calcium salt of inositol.
The phosphate moieties of phytic acid chelates divalent and trivalent cations such as metal ions, i.a. the nutritionally essential ions of calcium, iron, zinc and magnesium as well as the trace minerals manganese, copper and molybdenum.
Phytic acid and its salts, phytates, are often not metabolized, i.e. neither the phosphorous thereof, nor the chelated metal ions are nutritionally available.
Accordingly, food and feed preparations need to be supplemented with inorganic phosphate and often also the nutritionally essential ions such as iron and calcium, must be supplemented.
Still further, the phytate phosphorus passes through the gastrointestinal tract of such animals and is excreted with the manure, resulting in an undesirable phosphate pollution of the environment resulting e.g. in eutrophication of the water environment and extensive growth of algae.
Phytic acid or phytates, said terms being, unless otherwise indicated, in the present context used synonymously or at random, are degradable by phytases.
The production of phytases by plants as well as by microorganisms has been reported. Amongst the microorganisms, phytase producing bacteria as well as phytase producing fungi are known.
There are several descriptions of phytase producing filamentous fungi belonging to the fungal phylum of Ascomycota (ascomycetes). In particular, there are several references to phytase producing ascomycetes of the Aspergillus genus such as Aspergillus terreus (Yamada et al., 1986, Agric. Biol. Chem. 322:1275-1282). Also, the cloning and expression of the phytase gene from Aspergillus niger var. awamori has been described (Piddington et al., 1993, Gene 133:55-62). EP 0420358 describes the cloning and expression of a phytase of Aspergillus ficuum (niger). EP 0684313 describes the cloning and expression of phytases of the ascomycetes Aspergillus niger, Myceliophthora thermophila, Aspergillus terreus. Still further, some partial sequences of phytases of Aspergillus nidulans, Talaromyces thermophilus, Aspergillus fumigatus and another strain of Aspergillus terreus are given.
The cloning and expression of a phytase of Thermomyces lanuginosus is described in WO 97/35017.
There is a current need for phytases of amended properties or characteristics, e.g. phytases of increased thermostability, altered pH optimum (a high pH optimum being desirable for in-vitro processing, a low for in-vivo processing in the gastro-intestinal tract), and/or of a higher specific activity.
In a first aspect, the invention provides phytase variants, the characteristics of which are amendedxe2x80x94as compared to a so-called model phytase.
Any model phytase, which is of a certain similarity to thirteen herein specifically disclosed model phytases, can be made the model of such variants.
In another aspect, the invention relates to a novel phytase derived from Cladorrhinum foecundissimum. 
In still another aspect, the invention provides DNA sequences encoding these phytase variants and this phytase, and methods of their production.
Finally, the invention also relates generally to the use of the phytase and the phytase variants for liberating phosphorous from any phytase substrate, in particular inorganic phosphate from phytate or phytic acid.